Calcium homeostasis plays an important role in lens transparency, opacification, and cataractogenesis. Cataracts can occur both under hypocalcemic and hypercalcemic conditions, so the actual amount of available calcium in the lens is an important parameter for the health of the lens (1, 2). The normal mammalian lens has around 0.2-mM total calcium, of which the amount of free Ca2+ is only of the order of a few micromolars. Thus there must exist calcium regulation systems in the lens, and it is of interest to identify what they are and how they change in health and in disease. Vrensen et al. (3) have done an ultrastructural analysis of calcium distribution in the rat lens and have found calcium precipitates in the intermediate cortex fiber membranes, cytoplasm, and the nuclear envelope and very low levels of calcium in gap junctions, epithelial cells, and superficial fibers (3-5). The question of what the calcium-binding and -storing agents are in the lens is open; phospholipids and crystallins have been thought of as candidates. The major components of the lens are cytosolic proteins, crystallins, which account for about 40% of the wet weight of the lens. It is worth investigating whether any of the crystallins could act as; calcium sponge or storage depot in the tissue, particularly sine the ultrastructural analysis shows calcium distribution in the cytoplasm. We have earlier shown that the beta and avian core protein delta-crystallins show significant calcium-binding ability; (6, 7). Thus, the possibility of crystallins acting as lenticular calcium-sequestering and -storing systems exists.
However the calcium binding properties of gamma-crystallin have not yet been reported.
Gamma-Crystallin is a well-studied protein and was the first crystallin whose structure was solved (8). The Greek key crystallin fold was first described in this protein (8). It was later found in another lens protein, beta-crystallin, and in several other non-lens proteins, which were together classified as the beta-gamma-crystallin superfamily (9,10). The crystallin fold, also called the beta-gamma motif is a super-secondary structure formed from the symmetrical association of the two Greek key motifs that are organized into a two four-stranded anti-parallel beta-sheets (8, 9). The crystallin fold is a protein domain in which aromatic residues Tyr/Phe/Trp at position 1 and Gly at position 8 constitute the conserved sequence (Y/F/W) XXXXXXG, followed by a Ser at positions 28-34 from the first Y/F/W residue, and this sequence is repeated within 40 residues. Gly-8 is irreplaceable and is needed for forming a dihedral angle, which is not possible with another amino acid. These residues are required for the stabilization of the folded hairpin of the beta-gamma motif (11). Between Gly-8 and Ser-34 lie two charge clusters of alternate signs (12).
More members have been added to the diverse beta-gamma-crystallin superfamily. Protein S, a development-specific protein from Myxococcus xanthus (13-15), spherulin 3a from Physarun polycephalum (16, 17), AIM1 (absent in melanoma) which is associated with the tumorigenicity in human malignant melanoma (18), epidermis differentiation-specific protein family; (EDSP or EP37) from the amphibian Cynops pyrrhogaster (19-21), a yeast killer toxin (WmKT) from Williopsis mrakii (22) Streptomyces metalloproteinase inhibitor (SMPI) (23), and the calmodulin-binding membrane protein family (PCM) from Paramecium tetraurelia (24) are the non-lens members of the beta-gamma crystallin superfamily. Beta-gamma-Crystallins are thought to have originated from a single domain ancestor by gene duplication and gene fusion (25). The beta-gamma motif is seen in single domain (spherulin 3a and WmKT), two domain (beta- and gamma-crystallins, protein S EP37, SMPI) as well as multidomain proteins (AIM1). Evolutionarily, these proteins are among the most long-lived globular proteins known, generally expressed under stressed, adverse conditions or in differentiating tissues.
Table 1
Amino acid sequence of the Greek key crystallin fold peptide (corresponding to the third Greek key of bovine gamma-crystallin) and its mutants were synthesized and studied for calcium binding. Peptide s3a is the peptide corresponding to the first Greek key motif of spherulin 3a. Bold letters indicate the mutation of the residues.
TABLE IPeptidesAmino acid sequenceSeq. ID No.g390RMRIYERDDFRGQMSEITDDCPSLQDRFHLTEVHSLNVLEGS1311g3a90RMRIYKRDDFRGQMSEITDDCPSLQDRFHLTKVHSLNVLEGS1312g3b90RMRIYERDDFRGQMSEITKKCPSLQDRFHLTEVHSLNVLEGS1313g3a14GEVFLYKHVNFQGDSWKVTGNVYDFRSVSGLNDVVSSVKVGPN564
It is believed that the crystallin domain evolved in proteins of extraordinary stability. Members of this superfamily have, therefore, been studied for their stability and architecture. An interesting feature of some of these proteins is their calcium-binding ability, e.g. beta-crystallin (6, 7, 26), protein S (14, 27), and spherulin 3a (17). Putative calcium-binding sites have been shown in the EP37 protein (20). However, these proteins do not have any of the well-characterized motifs for calcium binding, such as the EF-hand, lipocortin-, or the annexin-like domains. They thus seem to contain an “orphan” motif, which needs to be identified.
Surprisingly, the calcium-binding ability of gamma-crystallin is not yet known, although it is the representative model of the superfamily, whose three-dimensional structure is very well known. Studying the calcium binding to gamma-crystallin not only points to the inherent characteristic of the superfamily but would also help in identifying the orphan motif in the members of beta-gamma-crystallin superfamily. In this work, we report that gamma-crystallin does bind calcium. We also show that the Greek key crystallin fold forms the calcium-binding site. Since members of this superfamily share a homologous crystallin fold, we suggest that other members also bind calcium and thus represent a novel class of calcium-binding proteins.